Not Applicable
This invention relates to colloidal particles of differing elemental compositions with differing morphologies and to methods of preparing same. The invention is particularly well-suited for use of the colloidal particles as labels in microscopy, as staining agents, and as biomolecule or drug carriers.
Colloidal particles have found myriad uses from biology to electronics. Colloidal gold, for example, has found particular use in biological labeling for localization of cell components at the microscopic and the ultrastructural level. In such use, colloidal gold is conjugated to a wide variety of biologically active molecules, i.e., biomolecules, such as antibodies, antibody fragments, ligands, ligand fragments or other molecular species that bind specifically to cellular targets. Colloidal gold particles can be made in sizes ranging from 1-150 nm. Depending on the particle size, colloidal gold is visible, or can be enhanced to be visible, in light microscopy, electron microscopy, x-ray microscopy or scanning force microscopy. Single particles of 10 nm and above are detectable but not resolvable by various types of light microscopy. Larger particles are most useful for applications where ease of detectability is important. Medium sized particles (10-20 nm) are still large enough for single particles to be detected by light microscopy (LM) and are resolvable by scanning electron microscopy (SEM) and transmission electron microscopy (TEM), with particles as small as xcx9c1 nm being detectable by TEM.
The use of colloidal gold particles as cellular markers in multiple labeling applications, however, is problematic. Multiple labeling which compares the localization of one type of label to another requires the use of different sized gold particles. The need to use differently sized particles, whose sizes do not overlap, limits the level of spatial resolution attainable as well as the total number of different cellular targets that can be labeled simultaneously. While smaller-sized particles permit a high degree of spatial resolution, the level of resolution drops rapidly with increasing particle size. This severely limits the precision of co-localization, particularly in the molecular and sub-molecular size ranges.
Thus, a problem, largely unattended in the art, is the lack of colloidal labels that are not dependent upon varying particle size to achieve co-localization and identification of multiple cellular targets. There is a need for smaller particles of differing elemental composition in cases where the path to the structure or molecule to be labeled is obscured by other structures, when the structures to be labeled are small and close together, or when a high particle concentration is necessary to achieve satisfactory labeling density.
The present invention provides a heretofore unmet need for colloidal particles of differing elemental composition with differing morphologies. Colloidal particles of differing shapes are suitably made of several different metals. Differing elemental compositions permit multiple labeling procedures with similarly sized particles. Resolution is thus possible with TEM, SEM in both secondary and backscattered modes, and with force microscopy in constant force, tapping and phase modes. The use of the colloidal particles of the present invention overcomes the limitations of prior art colloidal gold multiple labeling techniques requiring differently sized particles whose sizes do not overlap in spatial resolution, limiting the total number of different cell targets that can be labeled simultaneously.
The foregoing, and other advantages of the present invention, are realized in one aspect thereof in a method of making nonspherical colloidal particles of a metal selected from the group consisting of silver, palladium, platinum, rhodium, molybdenum and ruthenium. The method includes reducing a solution of a compound of the metal with a reducing agent selected from the group consisting of tannic acid, sodium citrate, ferrous sulfate, ascorbic acid, sodium borohydride, hydrogen, ethanol, methanol and combinations thereof. The colloidal particles made from the method of the present invention have morphologies or shapes which are cuboidal, lobate or geodesic. The particles range in size from about 1 nm to about 500 nm, preferably about 3 nm to about 18 nm.
In another aspect, the present invention provides a method of multi-immunolabeling or multiligand labeling which comprises conjugating to each biomolecule sought to be labeled metal colloidal particles in which the colloidal particles for each biomolecule differ in elemental composition or in morphology or a combination of both. The biomolecules are suitably an antibody, a cell, a cellular component, a cellular surface molecule, a protein, an antigen, a nucleic acid or fragment thereof or a drug. The colloidal particles are suitably selected from silver, palladium, platinum, rhodium, molybdenum and ruthenium and have shapes selected from spherical, cuboidal, lobate or geodesic.
In a further aspect, the present invention provides a method of gel staining which comprises staining each species sought to be stained with colloidal metal particles wherein the colloidal particles for each species differ in metal composition and/or particles size. The metals are selected from silver, palladium, platinum, rhodium, molybdenum and ruthenium, and the particle size ranges from about 1 nm to about 100 nm. It has been found that, depending on particle size, platinum and palladium particles are dark amber to golden-brown. Silver particles range in color from red to purple while, rhodium is amber or reddish brown to yellow, and ruthenium particles are orange to yellow orange and molybdenum is blue.
In yet another aspect, the present invention provides an in vivo delivery system, which system includes a biomolecule-carrier particle conjugate for delivering a biomolecule into the body of a subject/host wherein the carrier particles are colloidal particles of a metal selected from gold, silver, palladium, platinum, rhodium, molybdenum and ruthenium, and have a particle size ranging from about 1 nm to about 80 nm; and an administration means for administering the conjugate to the subject/host. The colloidal particles suitably have a morphology which is cuboidal, lobate or geodesic. The biomolecule is suitably a protein, a peptide, an antibody, a nucleic acid or fragments thereof, or a drug or other theraputic substance intended for use in diagnosis, treatment or prevention of a pathological condition.
Other advantages and a fuller appreciation of specific adaptations, compositional variations, and physical attributes will be gained upon an examination of the following detailed description of preferred embodiments, and the appended claims taken in conjunction with the figures of the drawing. It is expressly understood that the drawings are for the purpose of illustration and description only, and are not intended as a definition of the limits of the invention.